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Androgen induction of urokinase gene expression in LNCaP cells is dependent on their interaction with the extracellular matrix
Cancer Letters 130 (1998) 121–126
Androgen induction of urokinase gene expression in LNCaP cells is dependent on their interaction with the extracellular matrix Srinivas N. Pentyala, Terry C. Whyard, Wayne C. Waltzer, Alan G. Meek, Yaacov Hod* Prostate Cancer Research Center, Departments of Urology and Radiation Oncology, School of Medicine, State University of New York at Stony Brook, Stony Brook, NY, 11794-8093, USA Received 10 December 1997; received in revised form 15 April 1998; accepted 20 April 1998
Abstract Urokinase-type plasminogen activator (uPA) plays a central role in tissue remodeling and cell invasion. In the present study, we examined the expression of uPA in the prostate cancer cell lines LNCaP, DU-145 and PC-3. In contrast to DU-145 and PC3, the androgen-responsive cell line LNCaP does not express uPA. However, seeding LNCaP cells on fibronectin-coated plates stimulated a low level of uPA expression which was further induced upon exposure of the cells to dihydrotestosterone (DHT). Concomitant with the expression of uPA, an androgen-regulated expression of uPA receptor (uPAR) was induced. These results suggest that the interaction of LNCaP cells with the extracellular matrix plays a dominant role in the androgen control of uPA and uPAR gene expression. 1998 Elsevier Science Ireland Ltd. All rights reserved Keywords: LNCaP cells; Fibronectin; Dihydrotestosterone; Urokinase plasminogen activator; Urokinase plasminogen activator receptor
1. Introduction Expression of urokinase-type plasminogen activator (uPA) has long been associated with growth, malignant transformation and cellular movement such as monocytic cell migration, ovulation, metastasis and angiogenesis (reviewed in Ref. [1]). uPA is a serine protease that triggers the conversion of plasminogen into plasmin via specific proteolytic cleavage [2]. Plasmin, also a serine protease, has a broad spe* Corresponding author. Tel.: +1 516 4443721; fax: +1 516 4447620; e-mail: [email protected]
cificity and degrades a variety of proteinous components of the extracellular matrix (ECM), including fibrin, fibronectin and laminin [3], thereby amplifying the effect of uPA. uPA is secreted as a free form and then binds to a specific uPA receptor (uPAR) on the cell surface [4]. It has been demonstrated that the secreted form of uPA is inactive and only the cellsurface bound uPA mediates cell migration through the ECM [5]. It is believed that binding of uPA to its receptor confines and concentrates the enzyme to needed sites, thus enhancing migration potential and tissue remodeling. This is supported by the findings that uPAR is mostly localized in fibroblasts to sites of
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focal contacts and to the leading edge of migrating monocytes [6]. The interaction of the ECM with a variety of cells has been shown to modulate different pathways of signal transduction and the expression of a variety of genes, thereby influencing the phenotypic expression of the cell (reviewed in Ref. [7]). For instance, cell interaction with ECM has been demonstrated to modulate the expression and distribution of uPA and uPAR in HT-1080 sarcoma cells and WI-38 lung fibroblasts [8] and that of vitronectin, another matrix-associated protein that binds to uPAR [9]. Increasing evidence suggests that the association of prostate carcinoma cells with the surrounding ECM facilitates their growth [10]. For instance, growth stimulation of prostate cancer cells was demonstrated with growth factor-free ECM derived from normal human tissues [11]. It was well established that the invasion of the ECM by the androgen-independent prostate cancer cell lines PC-3 and DU-145 was contingent on endogenous uPA being bound to a specific cell surface receptor [12,13]. Neither uPA nor uPAR was detected in the androgen-responsive prostate cell line LNCaP, despite the fact that this cell line originates from a metastatic lesion [14]. The present study re-evaluates the expression of uPA and uPAR in LNCaP cells. We report here that androgen regulation of uPA and uPAR expression in LNCaP cells requires the interaction of the cells with components of the ECM. These results may have important implications in understanding the pathophysiological process of prostate cancer.
2. Materials and methods 2.1. Cell culture The human prostatic adenocarcinoma cell lines LNCaP, DU-145 and PC-3 were obtained from American Type Culture Collection (Rockville, MD) and maintained under standard conditions in the presence of RPMI 1640 (Bio-Whittaker) containing 10% Fetal Clone II calf serum (HyClone). Experiments were carried out with cell cultures at about 80% confluency and after the growth medium was replaced with serum-free media for 48 h. Cells were seeded on either plastic or fibronectin-coated plates as indicated in the
figure legends. Cell culture dishes were freshly coated with human plasma fibronectin (2.5 mg/cm2) for 45 min at room temperature as recommended by the supplier (Boehringer-Mannheim). The fibronectin solution was removed before cell seeding. 2.2. Measurement of uPA activity by zymography (casein underlay) uPA secreted to the medium was determined by zymography as described previously [15]. Briefly, media containing 10–50 mg of secreted proteins along side a purified uPA marker (American Diagnostica, CT) were subjected to SDS–polyacrylamide gel electrophoresis under standard conditions. The unstained gels were washed once with 2.5% Triton X-100 for 1 h, followed by a 1-h wash with distilled water to remove SDS. The gel was incubated at 37°C over an underlay bed consisting of 1% agarose, 50 mg/ ml human plasminogen and 5% Carnation non-fat milk (containing casein) in phosphate-buffered saline (PBS) until a clear zone of lysis appeared in the casein underlay (about 18–24 h). The uPA activates the underlying plasminogen, which, in turn, cleaves the casein in the milk, exposing a zone of lysis at the electrophoretic position of uPA. 2.3. Immunoblotting analysis of uPAR Cells were washed twice with cold PBS and then lysed in medium containing 10 mM Tris–HCl (pH 7.5), 1 mM EDTA, 10 mM MgCl2, 1% SDS, 0.25 M sucrose, 5 mg/ml leupeptin and 1 mM phenylmethylsulfonyl fluoride. Total cellular proteins (50 mg) were subjected to analysis by SDS–polyacrylamide gel electrophoresis as described above. Resolved proteins were electroblotted to Immobilon PVDF membrane (Millipore) using a Genie Transfer apparatus (Idea Scientific) at 24 V for 45 min with transfer buffer consisting of 25 mM Tris–base, 192 mM glycine and 20% methanol. After transfer, the membrane was blocked with non-fat milk (5%) suspended in 10 mM Tris–HCl (pH 7.5) and 150 mM NaCl (TBS) for 1 h and then incubated with rabbit antihuman uPAR antibody (American Diagnostica, CT) in TBS containing 1% albumin at room temperature for 1 h. Membranes were washed with TBS containing 0.2% Tween-20 and then incubated with horseradish
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peroxidase conjugate in a solution of 1% albumin in TBS for 45 min and uPAR was visualized using enhanced chemiluminescence (Amersham).
3. Results and discussion We first examined the expression of uPA in the three prostate cancer cell lines, LNCaP, PC-3 and DU-145. All three cell lines are metastatic and were isolated from lesions in lymph node, bone and brain, respectively. Out of the three, only LNCaP is androgen-responsive [16]. uPA was measured by zymography in which enzymatic activity was monitored by the capacity of uPA to activate plasminogen in an underlay as detailed in Section 2. uPA is secreted from the cell into the medium as a pro-enzyme, but it is activated by plasmin, as this enzyme is generated by the proteolytic conversion of plasminogen [17]. In the experiment shown in Fig. 1, LNCaP, PC-3 and DU145 cells were seeded on plastic plates under standard conditions. As seen, significant levels of uPA activity were noted with PC-3 and DU-145 cells, but no activity was detected in the medium bathing LNCaP cells for 48 h. uPA activity was detected in the incubation medium from PC-3 and DU-145 as early as after 16 h (data not shown). These results are consistent with an earlier study [14] that also did not find uPA activity in LNCaP cells. We ruled out the possibility that LNCaP cells secrete an inhibitor to uPA by examining its
Fig. 1. Differential expression of uPA in prostate cancer cell lines. DU-145, PC-3 and LNCaP cells were plated onto plastic plates (3 × 10−5 cells) and maintained in the presence of complete growth medium for 3 days. Two days after replacement into serum-free medium, aliquots of the incubation medium of DU-145 (lane 1), PC-3 (lane 2) and LNCaP (lane 3) containing 50 mg of protein were analyzed for uPA activity by casein underlay zymography as described in Section 2.
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activity in a mixed medium from LNCaP and PC-3 cells (data not shown). Furthermore, in zymography, the proteins are first denatured and separated by electrophoresis on polyacrylamide gel before the activity of uPA is measured (see Section 2). We next sought to determine whether the expression of uPA is arrested in LNCaP cells and whether it can be activated under suitable conditions. Because earlier studies have shown that components of the ECM can induce the expression of other matrix proteinases, such as matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) [18], we suspected that seeding LNCaP cells on an ECM may stimulate the expression of the uPA gene. Furthermore, Khan and Falcone [19] showed that seeding macrophages on specific components of the ECM stimulated the expression of uPA and MMP-9 and enhanced plasminogen activation. Therefore, we examined uPA activity in the medium of LNCaP cells seeded on fibronectin-coated plates. Fibronectin is one of the major components of the ECM to which cells anchor through members of cell surface integrins. This interaction is highly specific and mediated by the sequence Arg-Gly-Asp [20]. As seen in Fig. 2, culturing of LNCaP cells on fibronectin-coated plates resulted in significant expression of uPA. UPA activity was detected as early as 24 h following exposure of the cells to a serum-free medium and it continued to accumulate for at least another 24 h. We concluded from this experiment that expression of uPA is dependent on the interaction of LNCaP with the ECM. To the best of our knowledge, this is the first demonstration of uPA expression in LNCaP cells. uPA synthesis and expression is known to be modulated by a variety of effector molecules, such as phorbol esters, growth factors, peptide and steroid hormones, retinoids and several uncharacterized molecules secreted by cultured cells (reviewed in Ref. [1]). We examined next whether uPA expression in LNCaP cells is regulated by an androgen hormone. Androgens are required for the development of prostate and are known to induce cell growth [21]. The effect of androgens is mediated through the androgen receptor which belongs to the steroid superfamily of ligand-dependent transcription factors [22]. In the experiment seen in Fig. 3, LNCaP cells seeded on fibronectin-coated plates were exposed to increasing concentrations of DHT. As seen, very high levels of
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Fig. 2. Induction of uPA expression in LNCaP cells seeded on fibronectin-coated plates. Time-course analysis of uPA activity in the incubation medium of LNCaP cells seeded on fibronectincoated plates. Following 3 days in complete growth medium, cells were washed and exposed to serum-free medium. Aliquots were collected at the indicated times and analyzed for uPA activity by casein underlay zymography as described in Section 2. All other details are as in .
uPA activity were detected in the medium from cells incubated with DHT at a concentration above 10 − 9 M. While the analysis in this experiment was carried out with a limited amount of the growth medium to accommodate the differences in the expression of uPA in cells exposed to the different concentrations of DHT, significant stimulation in uPA activity was noted also at more physiological concentrations of the hormone (10 − 10 –10 − 11 M). In contrast, no uPA activity was detected in the media from cells seeded on plastic plates, even in the presence of a high concentration of DHT (10 − 8 M). While the cell numbers of cultures grown on Fn-coated dishes were two- to three-fold higher than those grown on uncoated dishes, uPA activity was undetectable in condition media collected from confluent cultures of LNCaP cells grown on plastic (data not shown), suggesting that uPA expression is not influenced by cell density. These results show that the interaction of LNCaP cells with the ECM makes them competent and responsive to extracellular stimuli of androgens. In contrast to PC-3 and DU-145 cells which express uPA irrespective of the extracellular environment, LNCaP cells seem to represent an intermediate step in tumor development; while these cells can metastasize when implanted in castrated animals [12], they still maintain responsiveness to components of the ECM. The exact mechanisms by which the ECM and DHT stimulate the expression of the uPA gene in LNCaP cells are not yet known. We suspect that DHT induces uPA gene transcription, as this seems to be the most common mechanism by which androgens regulate gene expression [23]. The mechanism mediated by the ECM is likely to be more complex and may involve the interaction of the ECM with
integrins, as demonstrated in monocytoid cells where the ECM modulated a6b1 and a6b4 integrin gene expression, and indirectly induce local proteolysis and cell migration [24]. uPA must be cell-surface bound in order to mediate cell migration through the matrix [6]. It is conceivable that in order for uPA to be active, the expression of uPA must be coordinated with the expression of its receptor (uPAR). Binding of uPA to its receptor activates uPA by an as yet unknown mechanism. Because the induction by DHT of uPA expression in LNCaP cells required the interaction of these cells with the ECM components (Fig. 2), we examined whether similar conditions are required to induce uPAR expression. A previous study identified uPAR in PC3 and DU-145 cells, but not in LNCaP cells, when plated under standard conditions [14]. We used Western blot analysis with antibody specific to uPAR to detect the receptor in LNCaP cells. As seen in Fig. 4, while DHT induced a low level of uPAR expression in cells grown under standard conditions (lanes 1 and 2), a marked increase in the level of the receptor was noted in cells grown on fibronectin-coated plates (lanes 3 and 4). These results show that the expression of uPA and uPAR is coordinately regulated by DHT in LNCaP cells. The results reported in the present study further emphasize the importance of the ECM in cell growth. Cell adhesion to components of the ECM is a requirement for cell growth and survival for a wide variety of cell types [25]. Cell adhesion to the ECM results in the activation of signaling pathways, which maintain cell
Fig. 3. DHT stimulates uPA gene expression. LNCaP cells seeded on fibronectin-coated plates as described in were exposed to serum-free medium containing the indicated concentrations of DHT. Aliquots of the medium containing 10 mg of protein were analyzed for uPA activity by casein underlay zymography as described in Section 2. Cells grown on plastic matrix following the same experimental regimen served as control. Pl, medium from LNCaP cells seeded on non-coated plastic plates; fibronectin, medium from cells seeded on fibronectin-coated plates.
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Fig. 4. Stimulation by DHT of uPAR gene expression in LNCaP cells seeded on fibronectin matrix. LNCaP cells were seeded on either plastic plates (lanes 1 and 2) or fibronectin-coated plates (lanes 3 and 4). The cells were incubated in serum-free medium with no further addition (lanes 1 and 3) or in the presence of DHT (10−9 M) (lanes 2 and 4) for 48 h. Western blot analysis was carried out as described in Section 2.
cycle progression from the G1 to the S phase. Anchorage of cells to the ECM is mediated to a large extent by integrins, a large family of heterodimeric cell surface receptors [26]. Integrin activation and ligation have been shown to activate mitogen-activated protein kinase (MAPK) via p21ras-dependent [27] and independent [28] pathways. Activation of MAPK, in turn, can regulate the transcription of other genes [29] and the translation of other mRNAs [30]. Although the present study does not establish the mechanism by which the ECM affects uPA and uPAR expression in LNCaP cells, our results are consistent with the notion that the interaction of cells with the ECM can modulate gene expression. LNCaP cells have been shown to be androgenresponsive even when seeded on plain uncoated dishes. For instance, incubation of LNCaP cells with DHT induced the expression of PSA [31], probasin [32] and other genes [33]. We confirmed these results by demonstrating that the level of probasin mRNA is stimulated upon exposure of LNCaP cells exposed to DHT (data not shown). These results suggest that the specific induction of uPA and uPAR in LNCaP requires some interaction between the pathways that mediate ECM and androgen signaling. The results reported in the present paper may explain the slow invasive ability of LNCaP cells relative to PC-3 and DU-145 cells. The expression of uPA/uPAR under the influence of androgen and matrix components implies a duality in function for this receptor that directly links a protease known to disrupt cell–matrix interactions with a matrix protein known to promote such interactions. This regulation of proteolytic activity at local environments should
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have important consequences for biological processes in which cells must degrade the ECM in order to invade and metastasize. Differences in the growth rate, morphology and functional gene expression of these cells under the influence of components of the ECM paves the way for better understanding of prostatic neoplasms and the design of new therapeutic models.
Acknowledgements We thank Drs James Quigley and John Sipley for helpful discussions during the course of this work.
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[22] R.M. Evans, The steroid and thyroid hormone receptor superfamily, Science 240 (1988) 889–895. [23] M.E. Taplin, G.J. Bubley, T.D. Shuster, M.E. Frantz, A.E. Spooner, G.K. Ogata, H.N. Keer, S.P. Balk, Mutation of the androgen-receptor gene in metastatic androgen-independent prostate cancer, N. Engl. J. Med. 332 (1995) 1393–1398. [24] A.E. Cress, I. Rabinovitz, W. Zhu, R.B. Nagle, The a6b1 and a6b4 integrins in human prostate cancer progression, Cancer Met. Rev. 14 (1995) 219–228. [25] S.M. Frisch, H. Francis, Disruption of epithelial cell–matrix interactions induces apoptosis, J. Cell Biol. 124 (1994) 619– 626. [26] M.A. Schwartz, M.D. Schaller, M.H. Ginsberg, Integrins: emerging paradigms of signal transduction, Annu. Rev. Cell. Dev. Biol. 11 (1995) 549–599. [27] E.A. Clark, R.O. Hynes, Ras activation is necessary for integrin-mediated activation of extracellular signal-regulated kinase 2 and cytosolic phospholipase A2 but not for cytoskeletal organization, J. Biol. Chem. 271 (1996) 14814–14818. [28] Q. Chen, T.H. Lin, C.J. Der, R.L. Juliano, Integrin-mediated activation of MEK and mitogen-activated protein kinase is independent of Ras, J. Biol. Chem. 271 (1996) 18122–18127. [29] A. Schulze, K. Zerfass-Thome, J. Berges, S. Middendorp, P. Jansen-Durr, B. Henglein, Anchorage-dependent transcription of Cyclin A gene, Mol. Cell. Biol. 16 (1996) 4632– 4638. [30] T.A. Lin, X. Kong, T.A. Haystead, A. Pause, G. Belsham, N. Sonenberg, J.C. Lawrence, PHAS-I as a link between mitogen-activated protein kinase and translation initiation, Science 266 (1994) 653–656. [31] C. Lee, D.M. Sutkowski, J.A. Sensibar, D. Zelner, I. Kim, I. Amsel, N. Shaw, G.S. Prins, J.M. Kozlowski, Regulation of proliferation and production of prostate-specific antigen in androgen-sensitive prostatic cancer cells, LNCaP, by DHT, Endocrinology 136 (1995) 796–803. [32] S. Kasper, P.S. Rennie, N. Bruchovsky, P.C. Sheppard, H. Cheng, L. Lin, R.P. Shiu, R. Snoek, R.J. Matusik, Cooperative binding of androgen receptors to two DNA sequences is required for androgen induction of the probasin gene, J. Biol. Chem. 269 (1994) 31763–31769. [33] X.H. Liu, H.S. Wiley, A.W. Meikle, Androgens regulate proliferation of human prostate cancer cells in culture by increasing transforming growth factor-alpha (TGF-alpha) and epidermal growth factor (EGF)/TGF-alpha receptor, J. Clin. Endocrinol. Metab. 77 (1993) 1472–1478.
Androgen induction of urokinase gene expression in LNCaP cells is dependent on their interaction with the extracellular matrix Srinivas N. Pentyala, Terry C. Whyard, Wayne C. Waltzer, Alan G. Meek, Yaacov Hod* Prostate Cancer Research Center, Departments of Urology and Radiation Oncology, School of Medicine, State University of New York at Stony Brook, Stony Brook, NY, 11794-8093, USA Received 10 December 1997; received in revised form 15 April 1998; accepted 20 April 1998
Abstract Urokinase-type plasminogen activator (uPA) plays a central role in tissue remodeling and cell invasion. In the present study, we examined the expression of uPA in the prostate cancer cell lines LNCaP, DU-145 and PC-3. In contrast to DU-145 and PC3, the androgen-responsive cell line LNCaP does not express uPA. However, seeding LNCaP cells on fibronectin-coated plates stimulated a low level of uPA expression which was further induced upon exposure of the cells to dihydrotestosterone (DHT). Concomitant with the expression of uPA, an androgen-regulated expression of uPA receptor (uPAR) was induced. These results suggest that the interaction of LNCaP cells with the extracellular matrix plays a dominant role in the androgen control of uPA and uPAR gene expression. 1998 Elsevier Science Ireland Ltd. All rights reserved Keywords: LNCaP cells; Fibronectin; Dihydrotestosterone; Urokinase plasminogen activator; Urokinase plasminogen activator receptor
1. Introduction Expression of urokinase-type plasminogen activator (uPA) has long been associated with growth, malignant transformation and cellular movement such as monocytic cell migration, ovulation, metastasis and angiogenesis (reviewed in Ref. [1]). uPA is a serine protease that triggers the conversion of plasminogen into plasmin via specific proteolytic cleavage [2]. Plasmin, also a serine protease, has a broad spe* Corresponding author. Tel.: +1 516 4443721; fax: +1 516 4447620; e-mail: [email protected]
cificity and degrades a variety of proteinous components of the extracellular matrix (ECM), including fibrin, fibronectin and laminin [3], thereby amplifying the effect of uPA. uPA is secreted as a free form and then binds to a specific uPA receptor (uPAR) on the cell surface [4]. It has been demonstrated that the secreted form of uPA is inactive and only the cellsurface bound uPA mediates cell migration through the ECM [5]. It is believed that binding of uPA to its receptor confines and concentrates the enzyme to needed sites, thus enhancing migration potential and tissue remodeling. This is supported by the findings that uPAR is mostly localized in fibroblasts to sites of
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focal contacts and to the leading edge of migrating monocytes [6]. The interaction of the ECM with a variety of cells has been shown to modulate different pathways of signal transduction and the expression of a variety of genes, thereby influencing the phenotypic expression of the cell (reviewed in Ref. [7]). For instance, cell interaction with ECM has been demonstrated to modulate the expression and distribution of uPA and uPAR in HT-1080 sarcoma cells and WI-38 lung fibroblasts [8] and that of vitronectin, another matrix-associated protein that binds to uPAR [9]. Increasing evidence suggests that the association of prostate carcinoma cells with the surrounding ECM facilitates their growth [10]. For instance, growth stimulation of prostate cancer cells was demonstrated with growth factor-free ECM derived from normal human tissues [11]. It was well established that the invasion of the ECM by the androgen-independent prostate cancer cell lines PC-3 and DU-145 was contingent on endogenous uPA being bound to a specific cell surface receptor [12,13]. Neither uPA nor uPAR was detected in the androgen-responsive prostate cell line LNCaP, despite the fact that this cell line originates from a metastatic lesion [14]. The present study re-evaluates the expression of uPA and uPAR in LNCaP cells. We report here that androgen regulation of uPA and uPAR expression in LNCaP cells requires the interaction of the cells with components of the ECM. These results may have important implications in understanding the pathophysiological process of prostate cancer.
2. Materials and methods 2.1. Cell culture The human prostatic adenocarcinoma cell lines LNCaP, DU-145 and PC-3 were obtained from American Type Culture Collection (Rockville, MD) and maintained under standard conditions in the presence of RPMI 1640 (Bio-Whittaker) containing 10% Fetal Clone II calf serum (HyClone). Experiments were carried out with cell cultures at about 80% confluency and after the growth medium was replaced with serum-free media for 48 h. Cells were seeded on either plastic or fibronectin-coated plates as indicated in the
figure legends. Cell culture dishes were freshly coated with human plasma fibronectin (2.5 mg/cm2) for 45 min at room temperature as recommended by the supplier (Boehringer-Mannheim). The fibronectin solution was removed before cell seeding. 2.2. Measurement of uPA activity by zymography (casein underlay) uPA secreted to the medium was determined by zymography as described previously [15]. Briefly, media containing 10–50 mg of secreted proteins along side a purified uPA marker (American Diagnostica, CT) were subjected to SDS–polyacrylamide gel electrophoresis under standard conditions. The unstained gels were washed once with 2.5% Triton X-100 for 1 h, followed by a 1-h wash with distilled water to remove SDS. The gel was incubated at 37°C over an underlay bed consisting of 1% agarose, 50 mg/ ml human plasminogen and 5% Carnation non-fat milk (containing casein) in phosphate-buffered saline (PBS) until a clear zone of lysis appeared in the casein underlay (about 18–24 h). The uPA activates the underlying plasminogen, which, in turn, cleaves the casein in the milk, exposing a zone of lysis at the electrophoretic position of uPA. 2.3. Immunoblotting analysis of uPAR Cells were washed twice with cold PBS and then lysed in medium containing 10 mM Tris–HCl (pH 7.5), 1 mM EDTA, 10 mM MgCl2, 1% SDS, 0.25 M sucrose, 5 mg/ml leupeptin and 1 mM phenylmethylsulfonyl fluoride. Total cellular proteins (50 mg) were subjected to analysis by SDS–polyacrylamide gel electrophoresis as described above. Resolved proteins were electroblotted to Immobilon PVDF membrane (Millipore) using a Genie Transfer apparatus (Idea Scientific) at 24 V for 45 min with transfer buffer consisting of 25 mM Tris–base, 192 mM glycine and 20% methanol. After transfer, the membrane was blocked with non-fat milk (5%) suspended in 10 mM Tris–HCl (pH 7.5) and 150 mM NaCl (TBS) for 1 h and then incubated with rabbit antihuman uPAR antibody (American Diagnostica, CT) in TBS containing 1% albumin at room temperature for 1 h. Membranes were washed with TBS containing 0.2% Tween-20 and then incubated with horseradish
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peroxidase conjugate in a solution of 1% albumin in TBS for 45 min and uPAR was visualized using enhanced chemiluminescence (Amersham).
3. Results and discussion We first examined the expression of uPA in the three prostate cancer cell lines, LNCaP, PC-3 and DU-145. All three cell lines are metastatic and were isolated from lesions in lymph node, bone and brain, respectively. Out of the three, only LNCaP is androgen-responsive [16]. uPA was measured by zymography in which enzymatic activity was monitored by the capacity of uPA to activate plasminogen in an underlay as detailed in Section 2. uPA is secreted from the cell into the medium as a pro-enzyme, but it is activated by plasmin, as this enzyme is generated by the proteolytic conversion of plasminogen [17]. In the experiment shown in Fig. 1, LNCaP, PC-3 and DU145 cells were seeded on plastic plates under standard conditions. As seen, significant levels of uPA activity were noted with PC-3 and DU-145 cells, but no activity was detected in the medium bathing LNCaP cells for 48 h. uPA activity was detected in the incubation medium from PC-3 and DU-145 as early as after 16 h (data not shown). These results are consistent with an earlier study [14] that also did not find uPA activity in LNCaP cells. We ruled out the possibility that LNCaP cells secrete an inhibitor to uPA by examining its
Fig. 1. Differential expression of uPA in prostate cancer cell lines. DU-145, PC-3 and LNCaP cells were plated onto plastic plates (3 × 10−5 cells) and maintained in the presence of complete growth medium for 3 days. Two days after replacement into serum-free medium, aliquots of the incubation medium of DU-145 (lane 1), PC-3 (lane 2) and LNCaP (lane 3) containing 50 mg of protein were analyzed for uPA activity by casein underlay zymography as described in Section 2.
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activity in a mixed medium from LNCaP and PC-3 cells (data not shown). Furthermore, in zymography, the proteins are first denatured and separated by electrophoresis on polyacrylamide gel before the activity of uPA is measured (see Section 2). We next sought to determine whether the expression of uPA is arrested in LNCaP cells and whether it can be activated under suitable conditions. Because earlier studies have shown that components of the ECM can induce the expression of other matrix proteinases, such as matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) [18], we suspected that seeding LNCaP cells on an ECM may stimulate the expression of the uPA gene. Furthermore, Khan and Falcone [19] showed that seeding macrophages on specific components of the ECM stimulated the expression of uPA and MMP-9 and enhanced plasminogen activation. Therefore, we examined uPA activity in the medium of LNCaP cells seeded on fibronectin-coated plates. Fibronectin is one of the major components of the ECM to which cells anchor through members of cell surface integrins. This interaction is highly specific and mediated by the sequence Arg-Gly-Asp [20]. As seen in Fig. 2, culturing of LNCaP cells on fibronectin-coated plates resulted in significant expression of uPA. UPA activity was detected as early as 24 h following exposure of the cells to a serum-free medium and it continued to accumulate for at least another 24 h. We concluded from this experiment that expression of uPA is dependent on the interaction of LNCaP with the ECM. To the best of our knowledge, this is the first demonstration of uPA expression in LNCaP cells. uPA synthesis and expression is known to be modulated by a variety of effector molecules, such as phorbol esters, growth factors, peptide and steroid hormones, retinoids and several uncharacterized molecules secreted by cultured cells (reviewed in Ref. [1]). We examined next whether uPA expression in LNCaP cells is regulated by an androgen hormone. Androgens are required for the development of prostate and are known to induce cell growth [21]. The effect of androgens is mediated through the androgen receptor which belongs to the steroid superfamily of ligand-dependent transcription factors [22]. In the experiment seen in Fig. 3, LNCaP cells seeded on fibronectin-coated plates were exposed to increasing concentrations of DHT. As seen, very high levels of
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Fig. 2. Induction of uPA expression in LNCaP cells seeded on fibronectin-coated plates. Time-course analysis of uPA activity in the incubation medium of LNCaP cells seeded on fibronectincoated plates. Following 3 days in complete growth medium, cells were washed and exposed to serum-free medium. Aliquots were collected at the indicated times and analyzed for uPA activity by casein underlay zymography as described in Section 2. All other details are as in .
uPA activity were detected in the medium from cells incubated with DHT at a concentration above 10 − 9 M. While the analysis in this experiment was carried out with a limited amount of the growth medium to accommodate the differences in the expression of uPA in cells exposed to the different concentrations of DHT, significant stimulation in uPA activity was noted also at more physiological concentrations of the hormone (10 − 10 –10 − 11 M). In contrast, no uPA activity was detected in the media from cells seeded on plastic plates, even in the presence of a high concentration of DHT (10 − 8 M). While the cell numbers of cultures grown on Fn-coated dishes were two- to three-fold higher than those grown on uncoated dishes, uPA activity was undetectable in condition media collected from confluent cultures of LNCaP cells grown on plastic (data not shown), suggesting that uPA expression is not influenced by cell density. These results show that the interaction of LNCaP cells with the ECM makes them competent and responsive to extracellular stimuli of androgens. In contrast to PC-3 and DU-145 cells which express uPA irrespective of the extracellular environment, LNCaP cells seem to represent an intermediate step in tumor development; while these cells can metastasize when implanted in castrated animals [12], they still maintain responsiveness to components of the ECM. The exact mechanisms by which the ECM and DHT stimulate the expression of the uPA gene in LNCaP cells are not yet known. We suspect that DHT induces uPA gene transcription, as this seems to be the most common mechanism by which androgens regulate gene expression [23]. The mechanism mediated by the ECM is likely to be more complex and may involve the interaction of the ECM with
integrins, as demonstrated in monocytoid cells where the ECM modulated a6b1 and a6b4 integrin gene expression, and indirectly induce local proteolysis and cell migration [24]. uPA must be cell-surface bound in order to mediate cell migration through the matrix [6]. It is conceivable that in order for uPA to be active, the expression of uPA must be coordinated with the expression of its receptor (uPAR). Binding of uPA to its receptor activates uPA by an as yet unknown mechanism. Because the induction by DHT of uPA expression in LNCaP cells required the interaction of these cells with the ECM components (Fig. 2), we examined whether similar conditions are required to induce uPAR expression. A previous study identified uPAR in PC3 and DU-145 cells, but not in LNCaP cells, when plated under standard conditions [14]. We used Western blot analysis with antibody specific to uPAR to detect the receptor in LNCaP cells. As seen in Fig. 4, while DHT induced a low level of uPAR expression in cells grown under standard conditions (lanes 1 and 2), a marked increase in the level of the receptor was noted in cells grown on fibronectin-coated plates (lanes 3 and 4). These results show that the expression of uPA and uPAR is coordinately regulated by DHT in LNCaP cells. The results reported in the present study further emphasize the importance of the ECM in cell growth. Cell adhesion to components of the ECM is a requirement for cell growth and survival for a wide variety of cell types [25]. Cell adhesion to the ECM results in the activation of signaling pathways, which maintain cell
Fig. 3. DHT stimulates uPA gene expression. LNCaP cells seeded on fibronectin-coated plates as described in were exposed to serum-free medium containing the indicated concentrations of DHT. Aliquots of the medium containing 10 mg of protein were analyzed for uPA activity by casein underlay zymography as described in Section 2. Cells grown on plastic matrix following the same experimental regimen served as control. Pl, medium from LNCaP cells seeded on non-coated plastic plates; fibronectin, medium from cells seeded on fibronectin-coated plates.
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Fig. 4. Stimulation by DHT of uPAR gene expression in LNCaP cells seeded on fibronectin matrix. LNCaP cells were seeded on either plastic plates (lanes 1 and 2) or fibronectin-coated plates (lanes 3 and 4). The cells were incubated in serum-free medium with no further addition (lanes 1 and 3) or in the presence of DHT (10−9 M) (lanes 2 and 4) for 48 h. Western blot analysis was carried out as described in Section 2.
cycle progression from the G1 to the S phase. Anchorage of cells to the ECM is mediated to a large extent by integrins, a large family of heterodimeric cell surface receptors [26]. Integrin activation and ligation have been shown to activate mitogen-activated protein kinase (MAPK) via p21ras-dependent [27] and independent [28] pathways. Activation of MAPK, in turn, can regulate the transcription of other genes [29] and the translation of other mRNAs [30]. Although the present study does not establish the mechanism by which the ECM affects uPA and uPAR expression in LNCaP cells, our results are consistent with the notion that the interaction of cells with the ECM can modulate gene expression. LNCaP cells have been shown to be androgenresponsive even when seeded on plain uncoated dishes. For instance, incubation of LNCaP cells with DHT induced the expression of PSA [31], probasin [32] and other genes [33]. We confirmed these results by demonstrating that the level of probasin mRNA is stimulated upon exposure of LNCaP cells exposed to DHT (data not shown). These results suggest that the specific induction of uPA and uPAR in LNCaP requires some interaction between the pathways that mediate ECM and androgen signaling. The results reported in the present paper may explain the slow invasive ability of LNCaP cells relative to PC-3 and DU-145 cells. The expression of uPA/uPAR under the influence of androgen and matrix components implies a duality in function for this receptor that directly links a protease known to disrupt cell–matrix interactions with a matrix protein known to promote such interactions. This regulation of proteolytic activity at local environments should
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have important consequences for biological processes in which cells must degrade the ECM in order to invade and metastasize. Differences in the growth rate, morphology and functional gene expression of these cells under the influence of components of the ECM paves the way for better understanding of prostatic neoplasms and the design of new therapeutic models.
Acknowledgements We thank Drs James Quigley and John Sipley for helpful discussions during the course of this work.
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